Objective To study the feasibil ity of preparation of the poly-D, L-lactide acid (PDLLA) scaffolds treated by ammonia plasma and subsequent conjugation of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides via amide l inkage formation. Methods PDLLA scaffolds (8 mm diameter, 1 mm thickness) were prepared by solvent casting/particulate leaching procedure and then treated by ammonia plasma. The consequent scaffolds were labeled as aminated PDLLA (A/ PDLLA). The pore size, porosity, and surface water contact angle of groups 0 (un-treated control), 5, 10, and 20 minutes A/ PDLLA were measured. A/PDLLA scaffolds in groups above were immersed into the FITC labelled GRGDS aqueous solutionwhich contain 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide(NHS), the molar ratio of peptides/EDC.HCL /NHS was 1.5 ∶ 1.5 ∶ 1.0, then brachytely sloshed for 24 hours in roomtemperature. The consequent scaffolds were labelled as peptides conjugated A/PDLLA (PA/PDLLA). The scaffolds in groups 0, 5, 10, and 20 minutes A/PDLLA and groups correspondingly conjugation of peptides were detected using X-ray photoelectron spectroscopy (XPS). The scaffolds in groups of conjugation of peptides were measured by confocal laser scanning microscope and high performance l iquid chromatography (HPLC), un-treated and un-conjugated scaffolds employed as control. Bone marrow mesenchymal stem cells (BMSCs) from SD rats were isolated and cultured by whole bone marrow adherent culture method. BMSCs at the 3rd–6th passages were seeded to the scaffolds as follows: 20 minutes ammonia plasma treatment (group A/PDLLA), 20 minutes ammonia plasma treatment and conjugation of GRGDS (group PA/PDLLA), and untreated PDLLA control (group PDLLA). After 16 hours of culture, the adhesive cells on scaffolds and the adhesive rate were calculated. After 4 and 8 days of culture, the BMSCs/scaffold composites was observed by scanning electron micorscope (SEM). Results No significant difference in pore size and porosity of PDLLA were observed between before and after ammonia plasma treatments (P gt; 0.05). With increased time of ammonia plasma treatment, the water contact angle of A/PDLLA scaffolds surface was decreased, and the hydrophil icity in the treated scaffolds was improved gradually, showing significant differences when these groups were compared with each other (P lt; 0.001). XPS results indicated that element nitrogen appeared on the surface of PDLLA treated by ammonia plasma. With time passing, the peak N1s became more visible, and the ratio of N/C increased more obviously. AfterPDLLA scaffolds treated for 0, 5, 10, and 20 minutes with ammonia plasma and subsequent conjugation of peptides, the ratio of N/C increased and the peak of S2p appeared on the surface. The confocal laser scanning microscope observation showed that the fluorescence intensity of PA/PDLLA scaffolds increased obviously with treatment time. The amount of peptides conjugated for 10 minutes and 20 minutes PA/PDLLA was detected by HPLC successfully, showing significant differences between 10 minutes and 20 minutes groups (P lt; 0.001). However, the amount of peptides conjugated in un-treated control and 0, 5 minutes PA/PDLLA scaffolds was too small to detect. After 16 hours co-culture of BMSCs/scaffolds, the adhesive cells and the adhesive rates of A/PDLLA and PA/PDLLA scaffolds were higher than those of PDLLA scaffolds, showing significant difference between every 2 groups (P lt; 0.01). Also, SEM observation confirmed that BMSCs proliferation in A/PDLLA and PA/PDLLA groups was more detectable than that in PDLLA group, especially in PA/PDLLA group. Conclusion Ammonia plasma treatment will significantly increase the amount of FITC-GRGDS peptides conjugated to surface of PDLLA via amide l inkage formation. This new type of biomimetic bone has stablized bioactivities and has proved to promote the adhesion and proliferation of BMSCs in PDLLA.
Objective To fabricate a nanohydroxyapatite-chitosan(nano-HA-CS) scaffold with high porosity by a simple and effective technique and to evaluate the physical and chemical properties and the cytocompatibility of the composite scaffold. Methods The threedimensional nano-HA-CS scaffolds with high porosity were prepared by the in situ hybridization-freeze-drying method. The microscopic morphology and components of the composite scaffolds were analyzed by the scanning electron microscopy (SEM), the transmission electron microscopy（TEM）, the X-ray diffraction（XRD）examination, and the Fourier transformed infrared spectroscopy（FTIR）. The calvarial osteoblasts were isolated from the neonatal Wistar rats. The serial subcultured cells (3rd passage) were respectively seeded onto the nanoHACS scaffold and the CS scaffold, and then were cocultured for 2, 4, 6 and 8 hours. At each time point,four specimens from each matrix were taken to determine the celladhesion rate. The cell morphology was observed by the histological staining and SEM. Results The macroporous nanoHACS scaffolds had a feature of high porosity with a pore diameter from 100 to 500 μm (mostly 400500 μm). The scaffolds had a high interval porosity; however, the interval porosity was obviously decreased and the scaffold density was increased with an increase in the contents of CS and HA. The SEM and TEM results showed that the nanosized HA was synthesized and was distributed on the pore walls homogeneously and continuously. The XRD and FTIR results showed that the HA crystals were carbonatesubstituded and not wellcrystallized. The cytocompatibility test showed that the seeded osteoblasts could adhere the scaffolds, proliferating and producing the extracellular matrix on the scaffolds. The adherence rate for the nanoHACS scaffolds was obviously higher than that for the pure CS scaffolds. Conclusion The nano-HA-CS scaffolds fabricated by the in situ hybridization-freeze-drying method have a good physical and chemical properties and a good cytocompatibility; therefore, this kind of scaffolds may be successfully used in the bone tissue engineering.
Objective To investigate the antioxidant and osteogenic induction capabilities of calcium phosphate nanoflowers (hereinafter referred to as nanoflowers) in vitro at different concentrations. Methods Nanoflowers were prepared using gelatin, tripolyphosphate, and calcium chloride. Their morphology, microstructure, elemental composition and distribution, diameter, and molecular constitution were characterized using scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and energy-dispersive spectroscopy. Femurs and tibias were harvested from twelve 4-week-old Sprague Dawley rats, and bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured using the whole bone marrow adherent method, followed by passaging. The third passage cells were identified as stem cells by flow cytometry and then co-cultured with nanoflowers at concentrations of 0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, and 3.6 mg/mL. Cell counting kit 8 (CCK-8) assay was performed to screen for the optimal concentration that demonstrated the best cell viability, which was subsequently used as the experimental concentration for further studies. After co-culturing BMSCs with the screened concentration of nanoflowers, the biocompatibility of the nanoflowers was verified through live/dead cell staining, scratch assay, and cytoskeleton staining. The antioxidant capacity was assessed by using reactive oxygen species (ROS) fluorescence staining. The in vitro osteoinductive ability was evaluated via alkaline phosphatase (ALP) staining, alizarin red staining, and immunofluorescence staining of osteocalcin (OCN) and Runt-related transcription factor 2 (RUNX2). All the above indicators were compared with the control group of normally cultured BMSCs without the addition of nanoflowers. Results Scanning electron microscopy revealed that the prepared nanoflowers exhibited a flower-like structure; transmission electron microscopy scans discovered that the nanoflowers possessed a multi-layered structure, and high-magnification images displayed continuous atomic arrangements, with the nanoflower diameter measuring (2.00±0.25) μm; energy-dispersive spectroscopy indicated that the nanoflowers contained elements such as C, N, O, P, and Ca, which were uniformly distributed across the flower region; Fourier transform infrared spectroscopy analyzed the absorption peaks of each component, demonstrating the successful preparation of the nanoflowers. Through CCK-8 screening, the concentrations of 0.8, 1.2, and 1.6 mg/mL were selected for subsequent experiments. The live/dead cell staining showed that nanoflowers at different concentrations exhibited good cell compatibility, with the 1.2 mg/mL concentration being the best (P<0.05). The scratch assay results indicated that the cell migration ability in the 1.2 mg/mL group was superior to the other groups (P<0.05). The cytoskeleton staining revealed that the cell morphology was well-extended in all concentration groups, with no significant difference compared to the control group. The ROS fluorescence staining demonstrated that the ROS fluorescence in all concentration groups decreased compared to the control group after lipopolysaccharide induction (P<0.05), with the 1.2 mg/mL group showing the weakest fluorescence. The ALP staining showed blue-purple nodular deposits around the cells in all groups, with the 1.2 mg/mL group being significantly more prominent. The alizarin red staining displayed orange-red mineralized nodules around the cells in all groups, with the 1.2 mg/mL group having more and denser nodules. The immunofluorescence staining revealed that the expressions of RUNX2 and OCN proteins in all concentration groups increased compared to the control group, with the 1.2 mg/mL group showing the strongest protein expression (P<0.05). Conclusion The study successfully prepares nanoflowers, among which the 1.2 mg/mL nanoflowers exhibits excellent cell compatibility, antioxidant properties, and osteogenic induction capability, demonstrating their potential as an artificial bone substitute material.
ObjectiveTo review the application and research progress of in vivo bioreactor as vascularization strategies in bone tissue engineering.
MethodsThe original articles about in vivo bioreactor that can enhance vascularization of tissue engineered bone were extensively reviewed and analyzed.
ResultsThe in vivo bioreactor can be created by periosteum, muscle, muscularis membrane, and fascia flap as well as biomaterials. Using in vivo bioreactor can effectively promote the establishment of a microcirculation in the tissue engineered bones, especially for large bone defects. However, main correlative researches, currently, are focused on animal experiments, more clinical trials will be carried out in the future.
ConclusionWith the rapid development of related technologies of bone tissue engineering, the use of in vivo bioreactor will to a large extent solve the bottleneck limitations and has the potential values for clinical application.
ObjectiveTo explore the effect of vascular endothelial growth factor 165 (VEGF165)-loaded porous poly (ε-caprolactone) (PCL) scaffolds on the osteogenic differentiation of adipose-derived stem cells (ADSCs).MethodsThe VEGF165-loaded porous PCL scaffolds (written, Sf-g/VEGF) were fabricated through a combination of solvent casting/salt leaching and a thermal-induced phase separation technique and then observed under scanning electron microscope (SEM). The release kinetics was determined by ELISA kit. The ADSCs were isolated from inguinal fat pads of 15 Sprague Dawley rats and cultured. The passage 3-4 ADSCs were seeded into the scaffolds, and then cultured in vitro for 7 days. The passage 3-4 ADSCs were seeded into the porous PCL scaffolds (written, Sf-g) as control. The alizarin red S (ARS) staining, ARS activity assay, and real-time quantitative PCR (RT-PCR) were performed to measure the osteogenic differentiation of ADSCs in vitro. Six Sprague Dawley rats were recruited to prepare the bilateral calvarial bone defects models (n=12). The 12 calvarial bone defects were randomly divided into 3 group (n=4). The defects of negative control group were not treated; the defects of Sf-g group and Sf-g/VEGF group were repaired with ADSCs-Sf-g scaffold complex and ADSCs-Sf-g scaffold complex, respectively. At 8 weeks after transplantation, the Micro-CT and HE staining were conducted to evaluate the osteogenic effects in vivo.ResultsThe morphology of the Sf-g/VEGF scaffolds were porous and well-connected, and the cumulative release rate was approximately 80% in 120 hours. The ARS staining showed that the ARS activity of Sf-g/VEGF group were stronger than that of Sf-g group (t=10.761, P=0.000). The mRNA expressions of osteogenic specific markers [special AT-rich sequence protein 2 (Satb2), alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN)] were significantly higher in Sf-g/VEGF group than in Sf-g group (P<0.05). The results of Micro-CT and HE staining also confirmed the promotion effect of Sf-g/VEGF scaffolds. All defects of 2 groups were partially repaired by new bone tissue, especially in Sf-g/VEGF group. The volume and area of new bone tissue were significantly higher in Sf-g/VEGF group than in Sf-g group (P<0.05).ConclusionThe VEGF165-loaded scaffolds can significantly improve the osteogenic differentiation of ADSCs both in vitro and in vivo.
Objective Tissue engineered bone implanted with sensory nerve can effectively promote angiogenesis and repair of bone defects. To investigate the effects of calcitonin gene-related peptide (CGRP) on proliferation and migration of human umbilical vein endothelial cells (HUVECs) for further revealing the mechanism of tissue engineered bone implanted with sensory nerve promoting angiogenesis. Methods HUVECs were collected from human umbilical core, and identified through von Willebrand factor (vWF) and CD31 immunofluorescence. The HUVECs were treated with CGRP and were ivided into 6 groups according to CGRP concentration: group A (0 mol/L), group B (1 × 10—12 mol/L), group C (1 × 10—11 mol/L), group D (1 × 10—10 mol/L), group E (1 × 10—9 mol/L), and group F (1 × 10—8 mol/L). The expression of the CGRP1 receptor (CGRP1R) was observed in HUVECs by cell immunofluorescence. The growth rate of HUVECs was detected through AlarmarBlue at 1, 2, 3, 4, and 5 days. Transwell chamber was used to detect the abil ity of cell migration. ELISA assay was used to detect the vascular endothel ial growth factor (VEGF) secretion and the protein expression of focal adhesion kinase (FAK) was examined using Western blot. Results HUVECs were identified through morphology, vWF and CD31 immunofluorescence. HUVECs expressed CGRP1R. CGRP could stimulate HUVECs prol iferation in a time- and concentration-dependent manners; the cell growth rates of groups B-F were significantly higher than that of group A at all time (P lt; 0.05); group F had highest cell growth rate. The number of cell migration of group B-F was significantly higher than that of group A (P lt; 0.05), which increased more than 3 times. Groups B-F had higher amount of VEGF than group A (P lt; 0.05), and groups C and D had highest amount of VEGF. FAK expression of groups B-F was significantly increased at 3, 7, and 10 days after CGRP treatment when compared with group A (P lt; 0.05). Conclusion CGRP may enhance the proliferation and migration of HUVECs by increasing the secretion of VEGF and expression of FAK.
Objective
To investigate the application potential of alginate-strontium (Sr) hydrogel as an injectable scaffold material in bone tissue engineering.
Methods
The alginate-Sr/-calcium (Ca) hydrogel beads were fabricated by adding 2.0wt% alginate sodium to 0.2 mol/L SrCl2/CaCl2 solution dropwise. Microstructure, modulus of compression, swelling rate, and degradability of alginate-Sr/-Ca hydrogels were tested. Bone marrow mesenchymal stem cells (BMSCs) were isolated from femoral bones of rabbits by flushing of marrow cavity. BMSCs at passage 5 were seeded onto the alginate-Sr hydrogel (experimental group) and alginate-Ca hydrogel (control group), and the viability and proliferation of BMSCs in 2 alginate hydrogels were assessed. The osteogenic differentiation of cells embeded in 2 alginate hydrogels was evaluated by alkaline phosphate (ALP) activity, osteoblast specific gene [Osterix (OSX), collagen type I, and Runx2] expression level and calcium deposition by fluorescent quantitative RT-PCR and alizarin red staining, Von Kossa staining. The BMSCs which were embeded in alginate-Ca hydrogel and cultured with common growth medium were harvested as blank control group.
Results
The micromorphology of alginate-Sr hydrogel was similar to that of the alginate-Ca hydrogel, with homogeneous pore structure; the modulus of compression of alginate-Sr hydrogel and alginate-Ca hydrogel was (186.53 ± 8.37) and (152.14 ± 7.45) kPa respectively, showing significant difference (t=6.853, P=0.002); there was no significant difference (t=0.737, P=0.502) in swelling rate between alginate-Sr hydrogel (14.32% ± 1.53%) and alginate-Ca hydrogel (15.25% ± 1.64%). The degradabilities of 2 alginate hydrogels were good; the degradation rate of alginate-Sr hydrogel was significantly lower than that of alginate-Ca hydrogel on the 20th, 25th, and 30th days (P lt; 0.05). At 1-4 days, the morphology of cells on 2 alginate hydrogels was spherical and then the shape was spindle or stellate. When three-dimensional cultured for 21 days, the DNA content of BMSCs in experimental group [(4.38 ± 0.24) g] was significantly higher than that in control group [(3.25 ± 0.21) g ] (t=8.108, P=0.001). On the 12th day after osteogenic differentiation, the ALP activity in experimental group was (15.28 ± 1.26) U/L, which was significantly higher than that in control group [(12.07 ± 1.12) U/L] (P lt; 0.05). Likewise, the mRNA expressions of OSX, collagen type I, and Runx2 in experimental group were significantly higher than those in control group (P lt; 0.05). On the 21th day after osteogenic differentiation, alizarin red staining and Von Kossa staining showed calcium deposition in 2 groups; the calcium nodules and phosphate deposition in experimental group were significantly higher than those in control group (P lt; 0.05).
Conclusion
Alginate-Sr hydrogel has good physicochemical properties and can promote the proliferation and osteogenic differentiation of BMSCs, so it is an excellent injectable scaffold material for bone tissue engineering.
ObjectiveTo investigate the feasibil ity of the domestic porous tantalum as scaffold material of bone tissue engineering by observing the expressions of osteogenesis related factors of MG63 cells co-cultured with domestic porous tantalum.
MethodsMG63 cells were cultured with porous tantalum scaffolds (group A), with porous tantalum leaching solution (group B), and with MEM as control group (group C). The cell adhesion of group A was observed on the scaffolds at 3, 5, and 7 days after culture by scanning electron microscopy (SEM); immunohistochemistry and Western blot methods were used to detect the expressions of Runt-related transcri ption factor 2 (Runx-2), osteocalcin (OC), and fibronectin (FN).
ResultsAt 3 days after culture, the cells of group A adhered the surface and pore of the porous tantalum scaffolds, with sparse cell arrangement and less protuberances; at 5 days after culture, adjacent cells connected to be a flat each other, which covered the surface and pore of the scaffold; at 7 days after culture, cells secreted plenty of extracellular matrix, covering most of the material surface. The expressions of Runx-2, OC, and FN were positive in 3 groups; darker staining of the cytoplasm was observed in group A, the expressions were significantly higher in group A than in other 2 groups. The results of immunohistochemistry and Western blot showed that the expressions of Runx-2 and OC were significantly increased in group A when compared with those in groups B and C (P < 0.05), but no significant difference was found between groups B and C (P > 0.05). The expression of FN had no significant difference among 3 groups (P > 0.05).
ConclusionDomestic porous tantalum could promote MG63 cells adhesion and growth, and may promote the expressions of Runx-2 and OC, so it can be used as a scaffold material of bone tissue engineering.
Objective To construct the recombinant adeno-associated virus vector with human bone morphogenetic protein 4 gene（AAV-hBMP4）. Methods The hBMP-4 gene primer was designed basing on the corresponding gene sequence in GenBank. EcoR I site was introduced into the upstream of the primer and Sal Ⅰ site into downstream. The hBMP-4 gene was amplifiedwith the template of EX-A0242-M01-hBMP-4, then was cloned into pUC18 vectorto construct recombinant plasmid pUC18-hBMP-4. The plasmids pUC18-hBMP-4 and plasmid pSNAV cut by EcoR Ⅰ and Sal Ⅰenzyme, the fragments were collected and linked with T4 DNA ligase at 16℃ over night, recombinant plasmid pSNAVhBMP-4 was obtained. The recombinant plasmid was then transfected into BHK21 cells using Lipofectamine TM2000. The G418 resistant cells were obtained consequently. Thesecells were infected with HSV1-rc/△UL2 which has the function of packaging andcopying the recombinant AAV. After purification, the construction of recombinant AAV-hBMP-4 was completed. Results The construction of the recombinant pSNAV-hBMP-4 was confirmed by PCR electrophoresis and digestion with restriction enzyme. The gene sequence in the recombinant pSNAV-hBMP-4 wascorrect. The virus titer was about 1.5×1012 μg/ml.The purity of the virus was more than 95% using the SDSPAGE method. Conclusion With this method, high virus titers and purity of AAV-hBMP-4 can be acquired successfully and it is useful to bone tissue engineering.
Objective To investigate the effects of flow shear stress and mass transport on the construction of largescale tissue engineered bone using a perfusion bioreactor. Methods Bone marrow (20 mL) was harvested from the il iac crestof the healthy volunteer, and then hBMSCs were isolated, cultured and identified. The hBMSCs at passage 3 were seeded on the critical-size β-TCP scaffold and cultured in a perfusion bioreactor for 28 days. Different flow shear stress (1 ×, 2 × and 3 ×) and different mass transport (3, 6 and 9 mL/min) were exerted on the cells seeded on the scaffold by changing the viscosity of media or perfusion flow rate. The cell prol iferation and ALP activity of cells seeded on the scaffold were detected, and histology observation and morphology measurement of cell/scaffold complex were conducted. Results When the perfusion flow rabe was 3 mL/min, the cell viabil ity of 2 × group was higher than that of other groups (P lt; 0.05). When the flow shear stress was 3 ×, no significant differences were found among 3, 6 and 9 mL/min in cell viabil ity (P gt; 0.05). When the perfusion flow rate was 3 mL/min, the activity of ALP of 2 × and 3 × groups was higher than that of 1 × group (P lt; 0.05). When the flow shear stress was 3 ×, the activity of ALP of 6 mL/min group was the highest (P lt; 0.05). After 28 days of perfusion culture, the ECM of all the groups distributed throughout the scaffold, and the formation and mineral ization of ECM was improved with the increase of flow shear stress when the perfusion flow rate was 3 mL/min. However, the increase of perfusion flow rate decreased the mineral ization of ECM when the flow shear stress was 3 ×. Conclusion As two important fluid dynamics parameters affecting the construction of large-scale tissue engineered bone, the flow shear stress and the mass transport should be measured duringthe process of constructing large-scale tissue engineered bone so as to maximize their roles.
